1. Field of the Invention
The present invention relates to DNA analysis and genetic diagnostics apparatuses such as an apparatus for sequencing the nucleotide sequence of DNA or identifying the types of nucleotides in DNA.
2. Background Art
Current methods for DNA sequencing used widely utilize gel electrophoresis and fluorescence detection. In this method, first, a large number of copies of a DNA fragment to be sequenced are prepared. Fluorescently labeled fragments with varying lengths starting at the 5′ end of the DNA are prepared. The fluorescent labels added to these DNA fragments differ in wavelength according to the type of a 3′-terminal nucleotide thereof. The difference by one nucleotide in length is distinguished by gel electrophoresis. Luminescence produced by each fragment group is detected. The types of the terminal nucleotides of the DNA fragment groups during measurement are determined based on their respective luminescence wavelengths. The DNA fragment groups pass, in order of increasing length, through a fluorescence detection part. Therefore, by measuring fluorescence color, the types of the terminal nucleotides of the DNA fragment groups can be determined in order of increasing length. As a result, the sequence of interest is determined. Such fluorescent DNA sequencers have been widely diffused and have played an important role in the human genome analysis (see Nature, Vol. 361, 565-566, 11 Feb. 1993). On the other hand, as announced in 2003, the humane genome sequencing has been completed. Now, sequence information is exploited in medical care or various industries. In these fields, long DNA does not have to be completely sequenced, and the determination of a short DNA sequence of interest is adequate in most cases. For this purpose, convenient and inexpensive apparatuses are required. Furthermore, the simultaneous sequencing of a very large number of DNA fragments has been increasingly demanded. For such DNA sequencing, convenient and extensible methods and apparatuses are required.
In response to these demands, techniques typified by pyrosequencing have been developed, which achieve sequencing through stepwise chemical reaction. In this method, primers are hybridized to a target DNA strand, and four nucleic acid substrates (dATP, dCTP, dGTP, and dTTP) for complementary strand synthesis are sequentially added one by one into a reaction solution where complementary strand synthesis reaction is in turn performed. Once the complementary strand synthesis reaction occurs, a DNA complementary strand extends to produce pyrophosphoric acid (PPi) as by-products. The pyrophosphoric acid is converted to ATP by the action of an enzyme coexisting therewith. This ATP reacts in the presence of luciferin and luciferase to produce luminescence. By detecting this light, the incorporation of the added substrate for complementary strand synthesis into the DNA strand is determined. As a result, the sequence information of the complementary strand, thus the sequence information of the target DNA strand, is determined (see Electrophoresis, 22, 3497-3504 (2001)). The original method of pyrosequencing was as follows (see U.S. Pat. No. 4,863,849): DNA was immobilized at some midpoint of a column, and a solution containing substrates for complementary strand synthesis was allowed to run in the column such that the reaction product pyrophosphoric acid passed through several reaction parts. In this process, the pyrophosphoric acid was converted to ATP, which then produced luminescence by use of a luciferin-luciferase luminescence system, and this luminescence was detected (see Analytical Biochemistry 174, 423-436 (1988)).
On the other hand, in a method disclosed by Nyren et al., substrates for complementary strand synthesis unused in the reaction were immediately degraded by use of an enzyme such as apyrase to eliminate the influence on next reaction step (see U.S. Pat. Nos. 4,971,903 and 6,258,568). This method may be achieved merely by sequentially adding reagents to a reaction chamber and is therefore more convenient. In the luciferin-luciferase luminescence system, not only ATP, but also dATP serving as a substrate for complementary strand synthesis, acts as a luminescent substrate. Thus, its analog dATPαS, which does not serve as a luminescent substrate, is used (see U.S. Pat. No. 6,210,891, JP Patent No. 3510272, and Analytical Biochemistry 242, 84-89 (1996)).
The present inventors have developed a method for highly sensitively examining a DNA sequence with less background luminescence, which comprises the process of producing ATP from pyrophosphoric acid and AMP using PPDK, instead of ATP sulfurylase conventionally used, as an enzyme involved in ATP-producing reaction from pyrophosphoric acid (see JP Patent Publication (Kokai) No. 2007-097471A (2007) and Analytical Chemistry, 78, 4482-4489, (2006)).
This method is also suitable for the parallel sequencing of many DNA samples. An attempt has been reported to sequence DNA samples in parallel by use of several tens of thousands to several millions of reaction cells (see WO2005/003375 and Nature, 437, 376-380 (2005)).